Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for gene therapy, regenerative medicine, screening of anticancer drugs and biotechnology. HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors. We previously constructed a synthetic HAC (tetO-HAC) with a conditional kinetochore. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, such as the tTS or tTA, to its centromeric tetO sequences. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the tetO-HAC. However, inactivation of the HAC kinetochore requires transfection of cells by a retrovirus vector to achieve a high level of tTS /tTA expression, a step that potentially may lead to insertional mutagenesis. In our recent work, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. We demonstrated that a single copy of tTAVP64 carrying 4 tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function. To adopt the alphoidtetO-HAC for routine gene function studies, we constructed a new TAR-BRV- tTAVP64 cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoidtetO-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells. In our previous studied, tetO-HAC was used to characterize protein/protein interaction in the functional kinetochore. In recent study, we exploited the HAC to study replication of human centromeres. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized in heterochromatin and centrochromatin occurred towards the end of the S phase. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, indicating that CENP-B has a role in regulating the replication of centromeric regions. We have also applied our tetO-HAC for screening of drugs affecting chromosome instability (CIN). Whole-chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a characteristic feature of most types of cancer, thus distinguishing them from their normal counterparts. Although CIN is generally considered a driver of tumor growth, a threshold level exists whereby further increase in CIN frequency becomes a barrier against tumor growth and therefore can be exploited therapeutically. However, drugs known to increase CIN beyond this therapeutic threshold are currently few in number. In our previous work, we have developed a new quantitative assay for measuring CIN based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene (Lee et al., 2013b). In 2015, we used this assay to rank 62 different anticancer drugs with respect to their effects on the fidelity of chromosome transmission. Included in this analysis were drugs with various mechanisms of action, including antimicrotubule drugs, histone deacetylase (HDAC) inhibitors, mitotic checkpoint inhibitors, and drugs that target DNA replication and DNA damage response. The drugs were ranked in order of their ability to induce HAC loss and the top ten drugs were paclitaxel, gemcitabine, dactylolide, LMP400, talazoparib, olaparib, peloruside A, GW843682, VX-680, and cisplatin. These compounds may be recommended as the first choice when CIN is considered as a target for cancer therapy. It is interesting that the top ten drugs include taxol, gemcitabine and cisplatin used as the front-line chemotherapeutic drugs for many types of cancer for decades. During the past year, we also worked to significantly increase the speed of our HAC-based CIN assay using a modified EGFP with a reduced half-life (e.g., EGFP with a degron box sequence). The new assay has been successfully applied for identification of genes controlling chromosome transmission. tetO-HAC represents a novel promising generation of high capacity episomal vectors. Its persistence, and any adverse effects, in various cell types in live animals, have not, however, been explored. In recent study we transferred the tetO-HAC into mouse ES cells and assessed whether the presence of this extra chromosome affects their pluripotent properties. tetO-HAC-bearing ES cells were indistinguishable from their wild-type counterparts: they retained self-renewal potential and full capacity for multilineage differentiation during mouse development, whereas the HAC itself was mitotically stable during this process. Our data provide the first example of synthetic HAC behaving like a normal chromosome in cells of living animals. It also opens a new perspective into functional genetic studies in laboratory animals as well as stem cell-based regenerative medicine. To address gene delivery and expression studies, during the past year we greatly improved efficiency of isolation of full-length human genes by Transformation-Associated Recombination (TAR) technique. TAR cloning in yeast has been developed in our lab for selective isolation of full-length genes and entire genomic loci from complex genomes. TAR cloning produces positive YAC clones with a desirable gene sequence at a frequency of 0.5%. We demonstrate that the yield of gene positive clones increases up to 40% after treatment of genomic DNA by Cas9 nucleases that cause two specific double strand breaks near the targeting sequences.